JP4712341B2 - Phospholipid membrane preparation - Google Patents

Description

  In the present invention, an antigen or an allergen is bound to the surface of a phospholipid membrane comprising a phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms and a phospholipid membrane stabilizer. The present invention relates to a phospholipid membrane preparation. Furthermore, it is related with the phospholipid membrane and phospholipid composition which are the raw materials.

  Substances that cause so-called allergic symptoms that are problematic in animals such as humans, livestock, and pets are those that exist mainly as environmental factors such as mite antigens, ragweed antigens, duck antigens, cedar pollen antigens, and eggs. Allergens present in foods such as milk, buckwheat, peanuts, fish or crustaceans are known. These allergic patients or livestock are very difficult to intentionally avoid exposure to allergens, and there is an urgent need to provide effective treatment or preventive medicine. The onset of allergy is considered to be caused by the production of IgE antibody, which is one of the antibodies. Therefore, it is considered that allergic symptoms can be treated or improved by suppressing the production of IgE antibody, or suppressing the production of IgE antibody and enhancing the production of IgG antibody.

  Hyposensitization therapy is known as one technique for the treatment and improvement of allergic diseases. The purpose of desensitization therapy is to induce remission of an immune response (IgE antibody production) by repeatedly administering a small amount of an allergen aqueous solution within a range where allergic symptoms can be tolerated. There are many problems in obtaining a certain effective effect by desensitization therapy, such as individual differences in immune response, the degree of allergic symptoms in patients or animals, and long-term treatment required. Moreover, since desensitization therapy is a passive therapy aimed at inducing remission of the immune response, there is a problem in widespread use as an effective therapy.

  As an improvement of this desensitization therapy, there is known a treatment method in which allergens are encapsulated in specific liposomes, and are orally or subcutaneously injected into allergic patients or patients to relieve allergic symptoms of patients or patients. . However, since this treatment method is also intended to make desensitization therapy safer, more effective and comfortable, it is a passive treatment method aiming at remission of immune response based on desensitization therapy. There is no change in a certain point. That is, it does not have an active function for regulating immune capacity, such as actively working on the production balance between IgG antibody and IgE antibody.

  Furthermore, as a method of using a liposome for treatment or improvement, a method of binding an allergen to a liposome using a known reactive phospholipid is also known. These technologies have been tackled for the purpose of suppressing the production of IgE antibody and enhancing the production of IgG antibody, but their immunoregulatory ability is insufficient, and the dosage is increased or the number of administration is increased. It does not have tolerable performance.

  These allergic symptom treatment improvement means are passive techniques (desensitization therapy) that gradually lowers or ameliorates allergen responsiveness, or aims to suppress IgE antibody production and enhance IgG antibody production However, this technology does not have sufficient effects. For this reason, it is a technique that requires intermittent and frequent administration or a large amount of administration, and is still insufficient for the widespread use of therapeutic actions. There is an urgent need to develop a preparation for allergy treatment and improvement that is sufficiently effective in practice and that can be used safely.

  Further, in animals such as humans, livestock, pets, and fish, various infectious diseases in which viruses or bacteria are pathogens are known as diseases. Vaccines are widely used to prevent such infections. As such a vaccine, an aluminum hydroxide mixed vaccine in which the antigen is made insoluble by adding aluminum hydroxide to the antigen solution, so-called precipitated vaccine, is often used. Specifically, precipitated diphtheria toxoid, precipitated diphtheria tetanus mixed toxoid, precipitated tetanus toxoid, precipitated hub poison toxoid, precipitated hepatitis B vaccine, precipitated purified pertussis vaccine, precipitated purified pertussis diphtheria tetanus mixed vaccine, etc. Has been.

  Usually, when a vaccine is inoculated, the living body acquires immunity and exhibits resistance by production of IgG antibodies or cellular immunity. However, when a vaccine is inoculated, an undesired biological immune response may occur, the most important of which is an allergic reaction (inoculation reaction) at the time of vaccination. This allergic reaction is mostly due to the production of excess IgE antibody, and erythema and swelling at the vaccination site, systemic shock symptom, etc. occur, and if the symptom is remarkable, it may lead to a life crisis. Thus, in addition to the main immune response such as the production of IgG antibodies that lead to infection prevention, allergic reactions occur as an unintended immune response, which is a major problem at the time of vaccination.

  Conventionally, as the main causative agent of allergic reaction, antigens used in vaccines themselves, components contained in antigens (for example, proteins in virus culture solution) or aluminum hydroxide gels used in the above-mentioned precipitated vaccines, etc. However, the causative substance has not been sufficiently identified yet. For this reason, although various techniques for suppressing IgE antibody production that causes an allergic reaction have been energetically studied, a technique having a practically sufficient effect has not yet been established.

  In recent years, the following techniques have been disclosed in such infectious disease prevention scenes. This is a technique in which an antigen is bound to a liposome surface using a known reactive phospholipid to enhance IgG antibody production and suppress IgE antibody production (Patent Document 1). However, this technique has a remarkably low immunoregulatory ability and is insufficient, and there are many problems in putting it to practical use, such as an increase in dose or an increase in the number of administrations. That is, comparing the vaccine using the aluminum hydroxide gel that has been put into practical use as an adjuvant with the liposomes in terms of immunity induction ability using the production amount of IgG antibody in the case of using the same antigen amount, the liposomes are vaccines. The effect is about 1/10 lower than that. Therefore, in order for the liposome to obtain the same effect as the vaccine, it is necessary to inject 10 times the amount or 10 times more frequent administration, and there are many problems for practical use.

  In addition, a liposome in which an antigen is bound on the surface by inserting a special hydrophobic peptide bound to a physiologically active substance or an antigen into the liposome membrane is known (Patent Document 2). This is a product in which various physiologically active substances are bound to the surface of a liposome. As an example close to application to a vaccine, the effect of enhancing the production of γ-INF, which is known as a cytokine that increases immunity, is mentioned. Yes. However, the above document does not specifically disclose that a technique in which a physiologically active substance is bound to the surface of a liposome using such a special peptide can be applied as a vaccine. In addition, the above-mentioned document has not yet studied specific suppression means for suppressing suppression of IgE antibody production and a practically sufficient enhancement effect of IgG antibody production. Furthermore, in this technique, there is a high risk that a special peptide is recognized as a foreign substance from a living body, induces IgE antibody production, and can induce a new allergic reaction. In addition, the peptide has poor compatibility with an acyl group or cholesterol that provides a hydrophobic environment in a phospholipid membrane such as a liposome, lacks stability as a preparation, or a physiologically active substance-binding peptide is released from the membrane, There is a concern that the effectiveness on the living body may change.

  Furthermore, an antigen-binding liposome in which a hydrophobic peptide bonded to an antigen is inserted into a liposome membrane is known (Patent Document 3). In this technique, a liposome composed of a phospholipid having a cholesterol of 10% or less and a fatty acid having 14 to 18 carbon atoms is used, and an antigen-bound peptide is inserted into a liposome membrane to obtain an antigen-bound liposome. Furthermore, using such liposomes, the survival rate of test animals is measured as an immunization test to evaluate the vaccine effect. Therefore, this liposome is not intended to provide a vaccine that is unlikely to cause an allergic reaction. In this technique, the hydrophobic domain of the peptide has 15 to 500 amino acids, and since phospholipids consisting of fatty acids having 14 to 18 carbon atoms are used, there is a risk that they will become unstable in the liposome membrane. There is sex. As described above, the above technique has many problems such as a risk of inducing an allergic reaction, lack of stability as a preparation, and a high possibility that the effectiveness on a living body is likely to change.

None of the above-described formulations using liposomes having antigens or physiologically active substances bound to the surface disclose vaccines that are unlikely to cause allergic reactions. Specifically, it has a sufficient ability to enhance the production of IgG antibodies practically, and furthermore, does not disclose the effect of suppressing the production of IgE antibodies. This is a technique that requires a large dose or is still insufficient for the widespread use of therapeutic actions. There is an urgent need to develop a vaccine preparation that is sufficiently effective in practice and that does not cause an allergic reaction that can be used safely.
JP 09-012480 A International Publication No. 02/098465 Pamphlet JP 2002-526436 A

  The present invention has been made in view of such circumstances, and the problem to be solved is an immune response characterized by suppressing the production of IgE antibodies and increasing the production of practically sufficient IgG antibodies. An object of the present invention is to provide a phospholipid membrane preparation that can be used for a vaccine that has an adjustment function and is less likely to cause allergic treatment or allergic reaction.

  As a result of intensive studies to solve the above problems, the present inventors have obtained a phospholipid membrane preparation in which an antigen or allergen is bound to the surface of a phospholipid membrane containing a specific phospholipid and a stabilizer. As a result, the inventors have obtained knowledge that the above problems can be solved, and have completed the present invention.

That is, the present invention is as follows.
(1) An antigen or allergen is bound to the surface of a phospholipid membrane containing a phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms and a phospholipid membrane stabilizer. A phospholipid membrane preparation characterized by the above.
(2) The phospholipid is at least one selected from diacylphosphatidylserine, diacylphosphatidylglycerol and diacylphosphatidic acid, diacylphosphatidylinositol, diacylphosphatidylcholine, diacylphosphatidylethanolamine, succinimidyl-diacylphosphatidylethanolamine, and maleimide-diacylphosphatidylethanolamine. The phospholipid membrane preparation according to (1) above, which is a seed.
(3) The phospholipid membrane preparation according to the above (1), wherein the phospholipid contains at least one selected from diacylphosphatidylserine, diacylphosphatidylglycerol and diacylphosphatidic acid.
(4) The phospholipid membrane preparation according to (1) above, comprising a lipid to which an antigen or allergen is bound by ionic bond or covalent bond.
(5) The phospholipid membrane preparation according to any one of (1) to (4) above, wherein the stabilizer is cholesterol.
(6) The phospholipid membrane preparation according to any one of (1) to (5) above, wherein the phospholipid membrane preparation is a liposome preparation.
(7) 1 to 99.6 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 0.2 to 75 mol% of a stabilizer for phospholipid membrane, and 10 to 12 carbon atoms A liposome preparation comprising 0.2 to 80 mol% of a lipid having an acyl group or a hydrocarbon group and having an antigen or allergen bound thereto.
(8) 1 to 99.5 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 0.2 to 75 mol% of a stabilizer for phospholipid membrane, and 10 to 12 carbon atoms 0.2 to 80 mol% of a lipid having an acyl group or hydrocarbon group and having an antigen or allergen bound thereto, and a neutral phospholipid 0.1 to 0.1 C having an acyl group or hydrocarbon group having 10 to 12 carbon atoms And a liposome preparation comprising 70 mol%.
(9) A phospholipid membrane comprising a phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, a reactive lipid, and a phospholipid membrane stabilizer.
(10) 1 to 99.6 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 0.2 to 75 mol% of a stabilizer for phospholipid membrane, and 10 to 12 carbon atoms And a reactive lipid having an acyl group or hydrocarbon group of 0.2 to 80 mol%.
(11) 1 to 99.5 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 0.2 to 75 mol% of a stabilizer for phospholipid membrane, and 10 to 12 carbon atoms Containing 0.2 to 80 mol% of a reactive lipid having an acyl group or hydrocarbon group and 0.1 to 70 mol% of a neutral phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms A phospholipid membrane characterized by comprising:

  According to the present invention, an antigen or allergen is bound to the surface of a phospholipid membrane comprising a phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms and a phospholipid membrane stabilizer. A phospholipid membrane preparation is provided. Such a phospholipid membrane preparation has an immune response regulating function that suppresses the production of IgE antibody and sufficiently increases the production of IgG antibody in practical use. For this reason, the phospholipid membrane preparation of the present invention can exhibit an excellent effect that allergic symptoms are unlikely to occur during vaccination for treatment / improvement of allergic diseases and prevention of infectious diseases. In addition, since the phospholipid membrane preparation of the present invention has a remarkably high effect of suppressing the production of IgE antibody and enhancing the production of IgG antibody, it can be a phospholipid membrane preparation containing a low concentration of antigen. Therefore, the phospholipid membrane preparation of the present invention is useful for vaccines used for treatment / amelioration of allergic diseases and prevention of infectious diseases.

Hereinafter, the present invention will be described in more detail.
The phospholipid membrane preparation of the present invention is a phospholipid membrane preparation for use in vaccines or allergy treatment, which has a C10-12 acyl group or hydrocarbon group, a phospholipid membrane stabilizer, An antigen or allergen is bound to the surface of a phospholipid membrane containing

  The phospholipid membrane used in the phospholipid membrane preparation of the present invention is composed of a phospholipid that is an amphiphilic surfactant, and this phospholipid forms an interface with the polar group facing the aqueous phase, and is hydrophobic. It has a structure in which the group faces the other side of the interface. Examples of the form of the phospholipid membrane include liposomes, phospholipid bilayer membranes, phospholipid micelles, and phospholipid emulsions. Here, the liposome has a double membrane structure of phospholipids having a closed space. The phospholipid bilayer membrane has an irregular two-layer structure. Phospholipid micelles and phospholipid emulsions have a single membrane structure of phospholipids. Among these, liposomes or phospholipid micelles are preferable, and liposomes are most preferable from the viewpoints of practicality, ease of formulation design, ease of production and quality control, and the like.

  The allergen used in the phospholipid membrane preparation of the present invention is not particularly limited as long as it is an allergen (allergic antigen) causing allergy. Examples of subjects that develop allergic symptoms include humans; pets such as dogs, cats, and small birds; and livestock such as chickens, ducks, pigs, cows, and sheep. Allergens include human and other animal allergens. Specific examples of these allergens include: house dust; mite antigen; ragweed antigen, camogaya antigen, cedar pollen antigen, mugwort antigen, canamgra antigen, chickpea antigen, etc .; grains such as rice, flour, buckwheat flour; milk, egg yolk, egg white Foods such as; epidermis such as dog hair, cat hair and feathers; and fungi such as Candida and Aspergillus. Among these, mite antigens, ragweed antigens, duck antigens, cedar pollen antigens, and egg white (OVA) antigens are preferably used in view of the number of allergic cases and the frequency of intake from foods. The allergens can be used alone or in combination of two or more.

  The antigen for a vaccine used in the phospholipid membrane preparation of the present invention may be any antigen that can be used as an antigen for a vaccine. Specifically, humans; pets such as dogs, cats, and small birds; chickens, ducks, and pigs Examples include substances that serve as antigens for livestock such as cattle and sheep. Examples of these antigens include various toxoids such as tetanus, diphtheria, and hub poison; influenza, polio, Japanese encephalitis, measles, mumps, rubella, rabies, yellow fever, chickenpox, hepatitis A, hepatitis B, hepatitis C , Nephrological hemorrhagic fever, proboscis fever, rotavirus infection, parvovirus, coronavirus, distemper virus, leptospira, infectious bronchitis virus, infectious leukemia virus, AIDS, etc .; cholera; BCG; malaria and the like. Of these antigens, human antigens or pet antigens, which are problematic for allergic reactions during vaccination, are preferred. Furthermore, antigens such as tetanus, diphtheria, Japanese encephalitis, polio, rubella, mumps in humans; parvovirus, coronavirus, distemper virus, leptospirer, infectious bronchitis virus, infectious leukemia virus in pets are more preferred. The above antigens can be used alone or in combination of two or more.

  Antigens or allergens are proteins, peptides, saccharides, and the like, and can be bound to the surface of the phospholipid membrane via their functional groups. Examples of such a functional group include an amino group, a thiol group, a carboxyl group, a hydroxyl group, a disulfide group, or a hydrophobic group composed of a hydrocarbon group having a methylene chain. Of these, amino groups, thiol groups, carboxyl groups, hydroxyl groups, and disulfide groups are covalent bonds, amino groups and carboxyl groups are ionic bonds, hydrophobic groups are hydrophobic bonds between hydrophobic groups, and antigens or allergens are phospholipid membranes. Can be bonded to the surface of Since antigens or allergens are often proteins or peptides, they have a high functional group content and are easy to put to practical use. From this viewpoint, the functional group possessed by the antigen or allergen is preferably an amino group, a carboxyl group, or a thiol group. In addition, when an antigen or allergen is saccharides, it is preferable to have a hydroxyl group as a functional group from the same viewpoint.

  In order to bind the phospholipid membrane to the functional group of the antigen or allergen, the phospholipid membrane is composed of an amino group, a succinimide group, a maleimide group, a thiol group, a carboxyl group, a hydroxyl group, a disulfide group, and a hydrocarbon group having a methylene chain. It preferably has a functional group such as a hydrophobic group. Since antigens or allergens are often proteins or peptides, amino groups, succinimide groups, and maleimide groups are preferred as the functional groups of the corresponding phospholipid membrane. The combination of the functional group possessed by the antigen or allergen and the functional group possessed by the phospholipid membrane can be freely selected within a range that does not affect the effect of the present invention. Preferred combinations include an amino group and an aldehyde, respectively. Group, amino group and amino group, amino group and succinimide group, thiol group and maleimide group. The ionic bond and the hydrophobic bond are preferable from the viewpoint of easy preparation of the phospholipid membrane preparation because the binding procedure of the antigen or allergen to the phospholipid membrane is simple. Further, the covalent bond is preferable from the viewpoint of the stability of binding of the antigen or allergen on the surface of the phospholipid membrane or the storage stability when the phospholipid membrane preparation is put into practical use. The phospholipid membrane preparation of the present invention is characterized in that an antigen or allergen is bound to the surface of the phospholipid membrane. As a result, in the practical stage, even after being administered into the living body by, for example, injection, the antigen or allergen is stably bound to the surface of the phospholipid membrane, so that the effect of the present invention can be further enhanced. From such a viewpoint, a covalent bond is preferable as the bond between the antigen or allergen and the phospholipid membrane.

  The phospholipid membrane used in the phospholipid membrane preparation of the present invention comprises a phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms and a phospholipid membrane stabilizer. The phospholipid used in the present invention has an acyl group or hydrocarbon group having 10 to 12 carbon atoms. Examples of the acyl group having 10 to 12 carbon atoms include a decanoyl group, an undecanoyl group, and a dodecanoyl group. Examples of the hydrocarbon group having 10 to 12 carbon atoms include a decyl group, an undecyl group, and a dodecyl group. As the phospholipid, glycerophospholipid can be preferably used. In this case, the acyl group or hydrocarbon group bonded to the 1-position and 2-position of the glycerin residue of the phospholipid may be the same or different. From the viewpoint of industrial productivity, the 1-position and the 2-position of the glycerin residue are preferably the same. Moreover, as a phospholipid, it is preferable to use the phospholipid which has a C10-C12 acyl group. In the present specification, phospholipid is a concept including a salt thereof. As the salt, a pharmacologically acceptable salt is preferable. Examples of such a salt include a salt of a phospholipid and an inorganic base, a salt of a phospholipid and an organic base such as ammonium. Examples of the salt with an inorganic base include alkali metal salts such as sodium, potassium and lithium, and alkaline earth metal salts such as calcium and magnesium. In these, alkali metal salts, such as a sodium salt, are suitable. In addition, the salt of phospholipid can be used 1 type or in combination of 2 or more types.

  An object of the present invention is to achieve an immune response regulating function characterized by suppressing production of IgE antibodies and increasing production of practically sufficient IgG antibodies. From the viewpoint of increasing production of practically sufficient IgG antibodies, the phospholipid preferably has an acyl group having 10 to 12 carbon atoms. When the number of carbon atoms of the acyl group exceeds 12, it is difficult to achieve sufficient production of IgG antibody. Moreover, when the carbon number of the acyl group is less than 10, the hydrophobic binding force of the hydrophobic group of the phospholipid is reduced, and the compatibility between the phospholipid and a phospholipid membrane stabilizer such as cholesterol is reduced, and the phospholipid membrane As a result, the stability is reduced. Examples of the phospholipid having an acyl group having 10 to 12 carbon atoms include acidic phospholipids, neutral phospholipids, and reactive phospholipids having a functional group capable of binding an antigen or allergen to the surface. These types and ratios are appropriately selected according to various requirements.

  As the acidic phospholipid, phospholipids such as phosphatidylserine, phosphatidylglycerol, phosphatidic acid, and phosphatidylinositol having an acyl group or hydrocarbon group having 10 to 12 carbon atoms can be used. From the viewpoints of industrial availability, quality for use as pharmaceuticals, and the like, diacylphosphatidylserine, diacylphosphatidylglycerol, diacylphosphatidic acid, and diacylphosphatidylinositol having an acyl group having 10 to 12 carbon atoms are preferable. Diacylphosphatidylserine and diacylphosphatidylglycerol are particularly preferable from the viewpoint of practically enhancing IgG antibody production, which is the subject of the present invention, industrial supplyability, quality for use as pharmaceuticals, and the like. Diacylphosphatidylserine is most preferred from the viewpoint of sufficiently enhancing production.

  Examples of the neutral phospholipid include phosphatidylcholine having an acyl group having 10 to 12 carbon atoms or a hydrocarbon group. The neutral phospholipid that can be used in the present invention is appropriately selected in the type and ratio within a range where the suppression of the production of IgE antibody and the increase in the production of a practically sufficient IgG antibody can be achieved. Can be used. Neutral phospholipids have a higher function of stabilizing phospholipid membranes than acidic phospholipids and lipids to which antigens or allergens are bound, and can improve membrane stability. From such a viewpoint, it is preferable that a neutral phospholipid is contained in the phospholipid membrane preparation of the present invention. Acid phospholipid, antigen- or allergen-bound lipid used to achieve the suppression of IgE antibody production and the practically sufficient increase in IgG antibody production, and the inclusion of a phospholipid membrane stabilizer The amount of neutral phospholipid used can be determined after securing the amount.

  In the phospholipid membrane preparation of the present invention, a phospholipid membrane preparation in which an antigen or allergen is bound to the surface of the phospholipid membrane can be obtained by containing a lipid to which an antigen or allergen is bound. In order to obtain a lipid to which this antigen or allergen is bound, a reactive lipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms can be used. Examples of the reactive lipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms include phospholipids having a reactive functional group, mono- or diacyl glycerides, fatty acids, and cationic lipids. The type and ratio of such reactive lipids can be appropriately selected according to various requirements. In addition, when carbon number of an acyl group or a hydrocarbon group exceeds 12, and when this carbon number is less than 10, it is unpreferable for the same reason as the above-mentioned.

  As the reactive lipid, a reactive phospholipid having an acyl group having 10 to 12 carbon atoms is preferably used in terms of stability in a phospholipid membrane, industrial supply, quality for use as a pharmaceutical product, and the like. . Examples of the reactive phospholipid include diacylphosphatidylethanolamine or a terminally modified product thereof. Moreover, diacyl phosphatidylglycerol, diacyl phosphatidylserine, diacyl phosphatidic acid, diacyl phosphatidylinositol, and these terminal modified bodies can also be used. From the viewpoint of industrial availability, simplicity of the binding step with an antigen or allergen, yield and the like, it is preferable to use diacylphosphatidylethanolamine or a terminally modified product thereof. Here, examples of the “terminally modified product” include a compound in which one end of a bifunctional reactive compound is bonded to the amino group of diacylphosphatidylethanolamine.

  Diacylphosphatidylethanolamine can be obtained, for example, by subjecting diacylphosphatidylcholine as a raw material to a base exchange reaction between choline and ethanolamine using phospholipase D. Specifically, first, a chloroform solution in which diacylphosphatidylcholine is dissolved and water in which phospholipase D and ethanolamine are dissolved are mixed at an appropriate ratio to obtain a crude reaction product. The crude reaction product can be purified on a silica gel column using a mixed solvent of chloroform / methanol / water to obtain the desired diacylphosphatidylethanolamine. Column purification conditions such as solvent composition ratio can be selected as appropriate.

  Examples of the bifunctional reactive compound include a compound having an aldehyde group or a succinimide group capable of reacting with the amino group of diacylphosphatidylethanolamine at at least one terminal. Specific examples of the bifunctional reactive compound having an aldehyde group include glyoxal, glutaraldehyde, succindialdehyde, terephthalaldehyde, and the like. Of these, glutaraldehyde is preferred. Specific examples of the bifunctional reactive compound having a succinimide group include dithiobis (succinimidyl propionate), ethylene glycol-bis (succinimidyl succinate), disuccinimidyl succinate, di Examples include succinimidyl suberate and disuccinimidyl glutarate.

  Examples of the bifunctional reactive compound having a succinimide group at one end and a maleimide group at the other end include N-succinimidyl-4- (p-maleimidophenyl) butyrate and sulfosuccinimidyl-4- (p -Maleimidophenyl) butyrate, N-succinimidyl-4- (p-maleimidophenyl) acetate, N-succinimidyl-4- (p-maleimidophenyl) propionate, succinimidyl-4- (N-maleimidoethyl) -cyclohexane-1-carboxyl Rate, sulfosuccinimidyl-4- (N-maleimidoethyl) -cyclohexane-1-carboxylate, N- (γ-maleimidobutyryloxy) succinimide, N- (ε-maleimidocaproyloxy) succinimide and the like. . When these reactive compounds are used, a diacylphosphatidylethanolamine terminal-modified product having a maleimide group is obtained. As described above, a diacylphosphatidylethanolamine terminal-modified product can be obtained by bonding a functional group at one end of a bifunctional reactive compound to the amino group of diacylphosphatidylethanolamine.

  As an example of the method of using the reactive lipid, a phospholipid composition containing the reactive lipid is prepared, and then the antigen or allergen is added to the antigen or allergen, followed by a predetermined treatment, whereby the antigen or allergen is bound. A phospholipid membrane preparation can be obtained. In addition, after binding an antigen or allergen to a reactive lipid in advance, this lipid is combined with a stabilizer of the phospholipid and the phospholipid membrane to prepare a phospholipid membrane, thereby binding the antigen or allergen. The inventive phospholipid membrane formulation may be prepared.

  In the present invention, sterols and tocopherols can be used as stabilizers for phospholipid membranes. As sterols, what is generally known as sterols may be used, and examples thereof include cholesterol, sitosterol, campesterol, stigmasterol, and brassicasterol. Among these, cholesterol is particularly preferable from the viewpoint of availability. Moreover, as tocopherols, what is generally known as tocopherol may be used, and commercially available α-tocopherol is preferable from the viewpoint of availability.

The phospholipid membrane preparation of the present invention preferably has the following composition.
1 to 99.6 mol% of phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 0.2 to 75 mol% of a stabilizer for phospholipid membrane, and an acyl group having 10 to 12 carbon atoms or A phospholipid membrane preparation comprising 0.2 to 80 mol% of a lipid having a hydrocarbon group and bound to an antigen or an allergen.

  The acidic phospholipid used in the present invention can be contained in an amount of 1 to 99.6 mol% with respect to all components of the phospholipid membrane. When the content is less than 1 mol%, the zeta potential is decreased, the stability of the phospholipid membrane is decreased, and it is difficult to sufficiently enhance IgG antibody production, which is the subject of the present invention. When the content exceeds 99.6 mol%, the content of the antigen or allergen-bound lipid and phospholipid membrane stabilizer necessary for achieving the object of the present invention becomes insufficient. From this viewpoint, the content of acidic phospholipid is preferably 2 to 90 mol%, more preferably 4 to 80 mol%, and most preferably 5 to 70 mol%.

  The content of neutral phospholipid is 0 to 80 mol%, preferably 0.1 to 70 mol%, more preferably 0.1 to 60 mol%, most preferably based on all components of the phospholipid membrane. Is 0.1 to 50 mol%. If it exceeds 80.0 mol%, it becomes difficult to sufficiently use acidic phospholipids, lipids bound with antigens or allergens and phospholipid membrane stabilizers necessary for achieving the object of the present invention.

  The lipid to which the antigen or allergen used in the present invention is bound can be contained in an amount of 0.2 to 80 mol% with respect to the total constituent components of the phospholipid membrane. If it is less than 0.2 mol%, the amount of antigen or allergen bound to the phospholipid membrane necessary to achieve practically sufficient enhancement of IgG antibody production will be insufficient. When it exceeds 80 mol%, the stability of the phospholipid membrane is lowered. The content of such lipid is preferably 1 to 55 mol%, more preferably 5 to 50 mol%.

  The reactive lipid used in the present invention can be contained in an amount of 0.2 to 80 mol% with respect to all components of the phospholipid membrane. If the amount is less than 0.2 mol%, a sufficient amount of antigen or allergen cannot be bound to the phospholipid membrane in order to achieve practically sufficient IgG antibody production enhancement. If it exceeds 80 mol%, the stability of the phospholipid membrane is lowered. From this viewpoint, the content of the reactive lipid is preferably 1 to 55 mol%, more preferably 5 to 50 mol%.

  The lipid to which the antigen or allergen used in the present invention is bound is obtained by binding the antigen or allergen to the reactive lipid described above. The ratio of the reactive lipid binding to the antigen or allergen can be appropriately selected from the types of functional groups used for binding, the binding treatment conditions, and the like within a range not impeding the effects of the present invention. For example, a terminal modified form of diacylphosphatidylethanolamine obtained by binding one end of disuccinimidyl succinate, which is a bifunctional reactive compound, to the terminal amino group of diacylphosphatidylethanolamine is used as a reactive phospholipid. When used, 10% or more, preferably 10 to 99% of the reactive phospholipid can be bound to the antigen or allergen by selection of various conditions such as binding treatment. In addition, the reactive phospholipid which is not couple | bonded with the antigen or the allergen becomes acidic phospholipid, and is contained in the phospholipid membrane formulation of this invention.

  The phospholipid membrane stabilizer that can be used in the present invention can be used in an amount of 0.2 to 75 mol% with respect to all components of the phospholipid membrane. From the standpoint of enhancing production of a practically sufficient IgG antibody that is the subject of the present invention, the content of the stabilizer is preferably as low as possible. However, from the point of suppressing production of IgE antibody, which is another subject, 0.2 mol % Or more is preferable. If it exceeds 75 mol%, the stability of the phospholipid membrane is impaired. The content of the stabilizer is preferably 1 to 70 mol%, more preferably 5 to 60 mol%, still more preferably 10 to 55 mol%.

Preferred embodiments of the phospholipid membrane preparation of the present invention include the following compositions. In addition, the composition shown below becomes a total of 100 mol%.
1 to 99.6 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 0.2 to 75 mol% of a stabilizer for phospholipid membrane, and an acyl group having 10 to 12 carbon atoms Alternatively, a phospholipid membrane preparation comprising 0.2 to 80 mol% of a lipid having a hydrocarbon group and bound with an antigen or allergen.
2 to 90 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 5 to 60 mol% of stabilizer for phospholipid membrane, and acyl group or hydrocarbon group having 10 to 12 carbon atoms A phospholipid membrane preparation comprising 1 to 55 mol% of a lipid having an antigen or an allergen bound thereto.
4 to 80 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 10 to 55 mol% of a stabilizer for phospholipid membrane, and an acyl group or hydrocarbon group having 10 to 12 carbon atoms And 5 to 50 mol% of a lipid to which an antigen or allergen is bound, and a phospholipid membrane preparation.

When the phospholipid membrane preparation contains neutral phospholipid, preferred embodiments include the following compositions.
1 to 99.6 mol% (preferably 1 to 99.5 mol%) of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, and 0.2 to 75 mol of a phospholipid membrane stabilizer %, A lipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms and bound with an antigen or allergen, and 0.1 to 70 mol% of neutral phospholipid. A phospholipid membrane preparation containing the composition.
2 to 90 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 5 to 60 mol% of stabilizer for phospholipid membrane, and acyl group or hydrocarbon group having 10 to 12 carbon atoms A phospholipid membrane preparation comprising 1 to 55 mol% of a lipid having an antigen or allergen bound thereto and 0.1 to 60 mol% of a neutral phospholipid.
4 to 80 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 10 to 55 mol% of a stabilizer for phospholipid membrane, and an acyl group or hydrocarbon group having 10 to 12 carbon atoms A phospholipid membrane preparation comprising 5 to 50 mol% of a lipid having an antigen or allergen bound thereto and 0.1 to 50 mol% of a neutral phospholipid.

When the acyl group or hydrocarbon group of the acidic phospholipid has 10 carbon atoms, the following composition is particularly preferable.
5 to 70 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 carbon atoms, 10 to 55 mol% of a stabilizer for phospholipid membrane, and an acyl group or hydrocarbon group having 10 to 12 carbon atoms A phospholipid membrane preparation comprising 5 to 50 mol% of a lipid to which an antigen or allergen is bound, and 0.1 to 50 mol% of a neutral phospholipid.

When the acyl group or hydrocarbon group of the acidic phospholipid has 10 carbon atoms, the following composition is more preferable.
A total of 5 to 40 mol% of diacylphosphatidylserine or diacylphosphatidylglycerol having an acyl group or hydrocarbon group having 10 carbon atoms, 10 to 55 mol% of a stabilizer for phospholipid membrane, an acyl group having 10 carbon atoms or carbonization A phospholipid membrane preparation comprising 5 to 50 mol% of a lipid having a hydrogen group and bound with an antigen or allergen, and 0.1 to 50 mol% of a neutral phospholipid.

In the case where the acyl group or the hydrocarbon group of the acidic phospholipid has 11 to 12 carbon atoms, the following composition is preferable.
5 to 70 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 11 to 12 carbon atoms, 10 to 55 mol% of a stabilizer for phospholipid membrane, and an acyl group or hydrocarbon group having 10 to 12 carbon atoms A phospholipid membrane preparation comprising 5 to 50 mol% of a lipid having an antigen or allergen bound thereto and 0 to 50 mol% of a neutral phospholipid.

In the case where the acyl group or the hydrocarbon group of the acidic phospholipid has 11 to 12 carbon atoms, the following composition is more preferable.
A total of 5 to 70 mol% of diacylphosphatidylserine or diacylphosphatidylglycerol having an acyl group or hydrocarbon group having 11 to 12 carbon atoms, 10 to 55 mol% of a phospholipid membrane stabilizer, and 11 to 12 carbon atoms A phospholipid membrane preparation comprising 5 to 50 mol% of a lipid having an acyl group or a hydrocarbon group and bound to an antigen or an allergen, and 0 to 50 mol% of a neutral phospholipid.

  In the present invention, the acyl group or hydrocarbon group contained in the lipid to which the phospholipid and the antigen or allergen are bound has 10 to 12 carbon atoms, but within the range not impeding the effects of the present invention. And compounds having less than 10 or more than 12 acyl groups or hydrocarbon groups may be included. For example, the ratio of the acyl group or hydrocarbon group having 10 to 12 carbon atoms is 25 mol% or more, preferably 50%, based on the total of all acyl groups or hydrocarbon groups contained in the phospholipid membrane preparation of the present invention. It is at least 75 mol%, more preferably at least 75 mol%, even more preferably at least 90 mol%, most preferably at least 97 mol%.

  Further, the phospholipid membrane preparation of the present invention is characterized by containing a phospholipid, and if it has 10 to 12 carbon atoms, it does not interfere with the effects of the present invention. Lipids other than phospholipids may be included. The content of such lipid is usually 40 mol% or less, preferably 20 mol% or less, more preferably 10 mol% or less.

  Furthermore, within the range which does not impair the effect of this invention, the structural component of the well-known phospholipid membrane which can comprise a phospholipid membrane may be included. The phospholipid membrane that can be used in the present invention is appropriately mixed and processed using phospholipids, reactive lipids, phospholipid membrane stabilizers, antigens or allergens, etc. as constituent components, and then these are appropriately used. It can be obtained by a method such as adding to a solvent. For example, when the phospholipid membrane is a liposome, the extrusion method, the vortex mixer method, the ultrasonic method, the surfactant removal method, the reverse phase evaporation method, the ethanol injection method, the prevesicle method, the French press method, W / O / It can be produced by a method such as a W emulsion method, an annealing method, or a freeze-thaw method. In addition, also when a phospholipid membrane is a phospholipid micelle, it can manufacture by the method similar to the above-mentioned.

  In the present invention, the form of the liposome is not particularly limited, but various sizes and forms such as multilamellar liposomes, small unilamellar liposomes, and large unilamellar liposomes can be obtained by appropriately selecting the aforementioned liposome production method. The liposome which has can be manufactured. In the present invention, the particle size of the liposome is not particularly limited, but from the viewpoint of storage stability, the particle size is preferably 20 to 600 nm, more preferably 30 to 500 nm, still more preferably 40 to 400 nm, particularly preferably. Is 50 to 300 nm, most preferably 70 to 230 nm. In addition, this particle size means an average particle size and can be measured by a dynamic light scattering method.

  In the present invention, in order to improve the physicochemical stability of the liposome, saccharides or polyhydric alcohols are added to the inner aqueous phase and / or the outer aqueous phase of the liposome after the liposome preparation process or adjustment. Also good. Especially when long-term storage or storage during formulation is necessary, after adding or dissolving sugar or polyhydric alcohol as a liposome protective agent, the water is removed by lyophilization and the lyophilized product of the phospholipid composition It is preferable to do.

  Examples of the saccharide include monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose and xylose; disaccharides such as saccharose, lactose, cellobiose, trehalose and maltose; trisaccharides such as raffinose and melezitose; Examples include oligosaccharides; polysaccharides such as dextrin; sugar alcohols such as xylitol, sorbitol, mannitol, and maltitol. Among these saccharides, monosaccharides or disaccharides are preferable, and glucose or saccharose is more preferable from the viewpoint of availability.

  Examples of polyhydric alcohols include glycerin compounds such as glycerin, diglycerin, triglycerin, tetraglycerin, pentaglycerin, hexaglycerin, heptaglycerin, octaglycerin, nonaglycerin, decaglycerin, polyglycerin; sorbitol, mannitol, etc. And the following sugar alcohol compounds: ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol, octaethylene glycol, nonaethylene glycol and the like. Among these, glycerin, diglycerin, triglycerin, sorbitol, mannitol, and polyethylene glycol having an average molecular weight of 400 to 10,000 are preferable from the viewpoint of availability. The concentration of saccharides or polyhydric alcohols contained in the inner aqueous phase and / or outer aqueous phase of the liposome is, for example, 1 to 20% by weight, preferably 2 to 10% by weight, based on the total weight of the liposome liquid. It is.

  When producing the phospholipid membrane preparation of the present invention, after producing the phospholipid membrane, the phospholipid membrane preparation can be easily obtained by binding an antigen or allergen to the surface of the phospholipid membrane. For example, a phospholipid membrane containing a phospholipid, a phospholipid membrane stabilizer, and a reactive lipid for binding an antigen or allergen to the membrane surface, such as a liposome solution, is prepared, and the outer aqueous phase is described above. About 2 to 10% by weight of sucrose, which is one of saccharides, is added and dissolved. The sugar-added preparation is transferred to a 10 ml glass vial, placed in a shelf-type freeze dryer, cooled to −40 ° C. or the like to freeze the sample, and a freeze-dried product is obtained by a conventional method. The freeze-dried product obtained here can be stored for a long period of time because the moisture has been removed. Moreover, the final phospholipid membrane preparation of the present invention can be easily and rapidly obtained by adding a specific antigen or allergen when necessary and carrying out the subsequent steps. When the interaction between the antigen or allergen and the phospholipid membrane is strong and the stability is low, etc., it is very important to store it at the lyophilized product of the phospholipid membrane and bind the antigen or allergen when necessary. Convenient.

Preferred embodiments of the phospholipid membrane of the present invention before reacting with an antigen or allergen are as follows. In addition, the composition shown below will be 100 mol% in total.
1 to 99.6 mol% of phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 0.2 to 75 mol% of a stabilizer for phospholipid membrane, and an acyl group having 10 to 12 carbon atoms or A phospholipid membrane comprising 0.2 to 80 mol% of a reactive lipid having a hydrocarbon group.

The following composition is mentioned as a preferable aspect of a phospholipid membrane.
1 to 99.6 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 0.2 to 75 mol% of a stabilizer for phospholipid membrane, and an acyl group having 10 to 12 carbon atoms Alternatively, a phospholipid membrane comprising 0.2 to 80 mol% of a reactive lipid having a hydrocarbon group.
2 to 90 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 5 to 60 mol% of stabilizer for phospholipid membrane, and acyl group or hydrocarbon group having 10 to 12 carbon atoms A phospholipid membrane comprising 1 to 55 mol% of reactive lipids having
4 to 80 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 10 to 55 mol% of a stabilizer for phospholipid membrane, and an acyl group or hydrocarbon group having 10 to 12 carbon atoms A phospholipid membrane comprising 5 to 50 mol% of a reactive lipid having a phospholipid.

When neutral phospholipid is contained as the phospholipid membrane, the following composition can be mentioned as a preferred embodiment.
1 to 99.6 mol% (preferably 1 to 99.5 mol%) of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, and 0.2 to 75 mol of a phospholipid membrane stabilizer , A phospholipid containing 0.2 to 80 mol% of a reactive lipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms and 0.1 to 70 mol% of a neutral phospholipid. film.
2 to 90 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 5 to 60 mol% of stabilizer for phospholipid membrane, and acyl group or hydrocarbon group having 10 to 12 carbon atoms A phospholipid membrane comprising 1 to 55 mol% of a reactive lipid having 0.1 and 0.1 to 60 mol% of a neutral phospholipid.
4 to 80 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 10 to 55 mol% of a stabilizer for phospholipid membrane, and an acyl group or hydrocarbon group having 10 to 12 carbon atoms A phospholipid membrane comprising 5 to 50 mol% of a reactive lipid having a neutral phospholipid content of 0.1 to 50 mol%.

In the case of containing an acidic phospholipid having 10 carbon atoms, the following composition is preferable.
5 to 70 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 carbon atoms, 10 to 55 mol% of a stabilizer for phospholipid membrane, and an acyl group or hydrocarbon group having 10 to 12 carbon atoms A phospholipid membrane comprising 5 to 50 mol% of reactive lipid and 0.1 to 50 mol% of neutral phospholipid.

In the case of containing an acidic phospholipid having 10 carbon atoms, the following composition is more preferable.
A total of 5 to 40 mol% of diacylphosphatidylserine or diacylphosphatidylglycerol having a C10 acyl group or hydrocarbon group, a phospholipid membrane stabilizer of 10 to 55 mol%, a C10 acyl group or carbonization A phospholipid membrane comprising 5 to 50 mol% of a reactive lipid having a hydrogen group and 0.1 to 50 mol% of a neutral phospholipid.

Moreover, when the C11-C12 acidic phospholipid is contained, the following composition is preferable.
5 to 70 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 11 to 12 carbon atoms, 10 to 55 mol% of a stabilizer for phospholipid membrane, and an acyl group or hydrocarbon group having 10 to 12 carbon atoms A phospholipid membrane comprising 5 to 50 mol% of a reactive lipid having 0 and 0 to 50 mol% of a neutral phospholipid.

Moreover, when the C11-12 acidic phospholipid is contained, the following composition is still more preferable.
A total of 5 to 70 mol% of diacylphosphatidylserine or diacylphosphatidylglycerol having an acyl group or hydrocarbon group having 11 to 12 carbon atoms, 10 to 55 mol% of a phospholipid membrane stabilizer, and 11 to 12 carbon atoms A phospholipid membrane comprising 5 to 50 mol% of a reactive lipid having an acyl group or a hydrocarbon group and 0 to 50 mol% of a neutral phospholipid.

The phospholipid membrane preparation of the present invention is characterized by containing a lipid to which an antigen or allergen is bound. Examples of a method for obtaining a phospholipid membrane preparation containing a lipid to which an antigen or allergen is bound include the following methods (A) and (B).
(A) After preparing a phospholipid membrane containing a phospholipid, a reactive lipid, and a phospholipid membrane stabilizer, an antigen or an allergen and a bifunctional reactive compound are added to the phospholipid membrane, In this method, the functional group of the reactive lipid contained in the membrane and the functional group of the antigen or allergen are bound via a bifunctional reactive compound. As a bifunctional reactive compound, the compound used when preparing the terminal modified body of a reactive lipid can be used similarly. Specific examples of the bifunctional reactive compound having an aldehyde group include glyoxal, glutaraldehyde, succindialdehyde, and terephthalaldehyde. Of these, glutaraldehyde is preferred. In addition, as a bifunctional reactive compound having a succinimide group, dithiobis (succinimidyl propionate), ethylene glycol-bis (succinimidyl succinate), disuccinimidyl succinate, disuccinimidyl Suberate, disuccinimidyl glutarate and the like can be used. Furthermore, as a bifunctional reactive compound having a succinimide group at one end and a maleimide group at the other end, N-succinimidyl-4- (p-maleimidophenyl) butyrate, sulfosuccinimidyl-4- (p- Maleimidophenyl) butyrate, N-succinimidyl-4- (p-maleimidophenyl) acetate, N-succinimidyl-4- (p-maleimidophenyl) propionate, succinimidyl-4- (N-maleimidoethyl) -cyclohexane-1-carboxylate , Sulfosuccinimidyl-4- (N-maleimidoethyl) -cyclohexane-1-carboxylate, N- (γ-maleimidobutyryloxy) succinimide, N- (ε-maleimidocaproyloxy) succinimide, etc. Can do. When such a bifunctional reactive compound is used, a diacylphosphatidylethanolamine terminal-modified product having a maleimide group as a functional group is obtained.
(B) After preparing a phospholipid membrane containing a phospholipid, a reactive lipid, and a phospholipid membrane stabilizer, an antigen or an allergen is added thereto, and the reactive lipid contained in the phospholipid membrane In this method, a functional group is bound to a functional group of an antigen or allergen.

  Examples of the type of the linking group in the above (A) and (B) include ionic bond, hydrophobic bond, and covalent bond. Furthermore, specific examples of the covalent bond include a Schiff base bond, an amide bond, a thioether bond, and an ester bond. In any of the above two methods, an antigen or allergen can be bound to the reactive lipid contained in the phospholipid membrane. Thereby, the lipid which the antigen or allergen couple | bonded in the phospholipid membrane is formed.

  In the method (A), as a specific example of the method of binding a phospholipid membrane as a raw material and an antigen or allergen with a bifunctional reactive compound, for example, a Schiff base bond can be used. . As a method of binding a phospholipid membrane and an antigen or allergen by Schiff base bonding, for example, a phospholipid membrane having an amino group on its surface is prepared to obtain a suspension of the phospholipid membrane, and the suspension of the phospholipid membrane. A method in which an antigen or allergen protein is added to a solution, a difunctional reactive compound dialdehyde is added, and the amino group on the surface of the phospholipid membrane is bound to the amino group of the antigen or allergen protein with a Schiff base. Can be mentioned.

As a specific example of this coupling procedure, for example, the following method may be mentioned.
(A-1) In order to obtain a phospholipid membrane having an amino group on the surface, diacylphosphatidylethanolamine having an acyl group or a hydrocarbon group having 10 to 12 carbon atoms is mixed into the raw lipid of the phospholipid membrane, A phospholipid membrane having a predetermined amount of amino groups on the surface of the phospholipid membrane is prepared.
(A-2) An antigen or allergen is added to the suspension of the phospholipid membrane.
(A-3) Next, glutaraldehyde is added as a bifunctional reactive compound and reacted for a predetermined time to form a Schiff base bond between the phospholipid membrane and the antigen or allergen.
(A-4) Thereafter, in order to deactivate excess glutaraldehyde, glycine as an amino group-containing water-soluble compound is added to the phospholipid membrane suspension and reacted.
(A-5) By removing the unbound antigen or allergen, the reaction product of glutaraldehyde and glycine, and excess glycine on the phospholipid membrane by a method such as gel filtration, dialysis, ultrafiltration, centrifugation, etc. A suspension of a phospholipid membrane having an antigen or allergen bound thereto is obtained.

  Specific examples of the method (B) include a method of introducing a reactive lipid having a functional group capable of forming an amide bond, a thioether bond, a Schiff base bond, an ester bond, or the like into a phospholipid membrane. Specific examples of such functional groups include succinimide group, maleimide group, amino group, imino group, carboxyl group, hydroxyl group, thiol group and the like. As an example of the reactive lipid introduced into the phospholipid membrane, a terminal modified product of the amino group terminal of diphosphatidylethanolamine having an acyl group or hydrocarbon group having 10 to 12 carbon atoms can be used.

As a specific example of this coupling procedure, for example, the following method may be mentioned.
(B-1) A diphosphatidylethanolamine having an acyl group or hydrocarbon group having 10 to 12 carbon atoms and one end of disuccinimidyl succinate are reacted by a known method, and one end is succinimide A disuccinimidyl succinate-linked diphosphatidylethanolamine having a group is obtained.
(B-2) Disuccinimidyl succinate-linked diphosphatidylethanolamine and other phospholipid membrane components are mixed by a known method to prepare a phospholipid membrane composition having a succinimide group on the surface.
(B-3) An antigen or allergen protein is added to the suspension of the phospholipid membrane composition, and the amino group of the antigen or allergen protein is reacted with the succinimide group on the surface of the phospholipid membrane.
(B-4) An unreacted antigen or allergen and a reaction replica are removed by a method such as gel filtration, dialysis, ultrafiltration, and centrifugation, and a phospholipid membrane containing a lipid to which the antigen or allergen is bound. A suspension is obtained.

  When binding a phospholipid membrane to an antigen or allergen, since the antigen or allergen is mainly a protein, it is practically preferable to target amino groups or thiol groups that are often contained as reactive groups. When an amino group is intended, it can be reacted with a succinimide group to obtain a Schiff base bond. When a thiol group is targeted, it can be reacted with a maleimide group to obtain a thioether bond. Since the phospholipid membrane preparation of the present invention includes a phospholipid membrane preparation in which an antigen or allergen is bound to the surface, it functions as a vaccine that hardly causes allergic treatment or allergic reaction. The posome preparation of the present invention can be used in routes such as oral, transdermal, transmucosal, subcutaneous, intravenous and intraperitoneal administration.

Next, the prescription and usage when the phospholipid membrane preparation of the present invention is used for allergy treatment will be described in detail.
As described above, the phospholipid membrane preparation of the present invention obtained by a method such as gel filtration, dialysis, ultrafiltration or centrifugation is a phospholipid membrane preparation in which an antigen or allergen is bound to the surface of the phospholipid membrane. Yes, in suspension. As the solvent for suspending, an aqueous solvent such as distilled water; physiological saline; phosphate buffer solution, carbonate buffer solution, Tris buffer solution, acetate buffer solution and the like can be used. The pH of such an aqueous solvent is 5 to 10, preferably 6 to 8. In addition, the above-described phospholipid membrane suspension can be pulverized by a process such as vacuum drying or freeze drying. And it can preserve | save in a powder state and can be used by disperse | distributing with the said aqueous solvent etc. at the time of use.

  The administration of the phospholipid membrane preparation of the present invention can be started at any time point when the allergic symptoms are present or before the allergic symptoms are exhibited. The degree of immune response of patients or livestock to allergens varies greatly depending on the living environment (environmental factors) or family (genetic factors) that have been contacted by nature. For this reason, it is preferable to determine the dosage of the phospholipid membrane formulation of this invention, an administration period, and the frequency | count of administration according to each individual of a patient or livestock. Specifically, by administering a phospholipid membrane preparation and tracking the IgE antibody titer against allergen in blood for a while, the degree of progress of allergy treatment can be grasped, and the dosage and administration period can be determined. . Administration frequency is, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months Can be implemented every time. In the case of patients or livestock with extremely sensitive allergic factors, it is preferable to continuously take the phospholipid membrane preparation of the present invention to improve the allergic constitution.

  The time when the phospholipid membrane preparation of the present invention is administered is a time before afflicted with an infectious disease, and a preferable time is a time after the time when the patient's maternal-derived transitional immunity decreases. Specifically, the preferred period is 2 years of age or later for humans, 3 weeks of age or later for dogs or cats, and 5 years of age or later for pigs or cattle. When a new and highly toxic infectious agent is generated, the phospholipid membrane preparation of the present invention can be ingested at the necessary time to exert the function of the vaccine. The dosage, administration period, and number of administrations of the phospholipid membrane preparation are affected by environmental factors or genetic factors similar to those described above, and the degree of immune response varies greatly depending on the individual of the patient or patient. It is preferable to determine accordingly. Specifically, the progress of vaccine therapy for the prevention of infectious diseases can be ascertained by administering a phospholipid membrane preparation and tracking the IgG antibody titer against the antigen in the blood for a while. The frequency of administration can be performed, for example, once a week, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, once a year or 2 years. Since tracking antibody titer can be very laborious, for example, the phospholipid membrane preparation of the present invention can be regularly administered to prevent infection.

  The phospholipid membrane preparation of the present invention is characterized by suppressing the production of IgE antibodies and enhancing the production of practically sufficient IgG antibodies when used as an allergy therapeutic agent or a vaccine for preventing infectious diseases. More specifically, the features of the present invention include IgE antibody (antibody titer after secondary immunization) / IgG antibody (secondary immunization) compared to any pharmaceutical preparation conventionally used in medical practice in the field. The ratio of antibody titer after immunization (hereinafter abbreviated as “IgE / IgG ratio”) is extremely low, and the antibody titer of IgG is sufficiently high. Compared to vaccines that use aluminum hydroxide gel as an adjuvant in many practical applications, the IgE / IgG ratio is remarkably low, and the IgG antibody titer is remarkably high in vaccines used for allergy treatment or infectious disease prevention. Very important and meaningful. The extremely low IgE / IgG ratio means that the degree of allergic symptoms caused by IgE antibodies is very low in vaccine therapy for allergy treatment and infection prevention. The remarkably high IgG antibody titer further contributes to lowering the IgE / IgG ratio in the scene of allergy treatment. In vaccine therapy for the prevention of infectious diseases, IgG antibodies are one of the main components of infectious disease preventing functions, and it is preferable that the antibody titer of IgG is higher.

  Since the phospholipid membrane preparation of the present invention can achieve remarkably high IgG antibody production, even if it is a preparation containing a lower concentration of antigen, the infection prevention function can be achieved. A vaccine preparation containing an antigen that is a foreign substance for a living body has an eternal problem of avoiding various side effects and adverse effects due to the antigen. In this regard, the phospholipid membrane preparation of the present invention, which has remarkably high IgG antibody production ability, is a very preferable technique because the antigen can be used at a low concentration level. Here, the above-mentioned “IgE / IgG ratio” means the ratio of titer when blood IgG antibody and IgE antibody are measured by ELISA. Even if the measurement methods of IgG antibody and IgE antibody are different, if the immunoregulatory ability is evaluated using the IgE / IgG ratio based on the same concept, production of IgG antibody that is practically sufficient can be suppressed by suppressing the production of IgE antibody. Can be confirmed to increase.

Hereinafter, the ELISA method used in the present invention will be described.
[ELISA method (IgG antibody detection)]
1) Coating of plate with antigen Antigen was dissolved in 0.05 M carbonate buffer (pH 9.0) at a concentration of 1 mg / ml, dispensed into a 96-well assay plate at 50 μl / well, and allowed to stand at room temperature for 1 hour. To do.
2) Blocking of the plate Bovine serum albumin (hereinafter referred to as “BSA”) was dissolved in 0.2 M phosphate buffer (pH 7.2: hereinafter referred to as “PBS”) at a concentration of 1 mg / ml. Dispense 100 μl / well into the plate and leave at room temperature for 1 hour.
3) Dilution and addition of serum sample (primary antibody) Immunization with PBS containing BSA at a concentration of 1 mg / ml (hereinafter referred to as “PBSA”) with aluminum hydroxide-antigen or the phospholipid membrane preparation of the present invention The mouse serum is diluted 11 times in a 2-fold step starting from a 10-fold dilution, dispensed into the plate of 2) at 50 μl / well and left at room temperature for 1 hour.
4) Addition of peroxidase-labeled rabbit anti-mouse IgG antibody solution (secondary antibody) After the plate of 3) above was washed three times with PBS, 50 μl / well of peroxidase-labeled rabbit anti-mouse IgG antibody in PBSA was dispensed. Leave at room temperature for 1 hour.
5) Addition of enzyme substrate solution After the plate of 4) above was washed 3 times with PBS, o-phenylenediamine dihydrochloride (0.5 mg / ml) dissolved in citrate buffer was dispensed at 100 μl / well. Let it develop at room temperature for 15 minutes. After color development, the reaction is stopped by dispensing 2 M sulfuric acid in a volume of 50 μl / well.
6) Measurement using an absorptiometer Measure the absorbance at 490 nm using an ELISA plate reader, determine the end point of color development, and use the average dilution factor of the sample serum at that point as the ELISA titer (ELISA titer Used as quantitative value of IgG antibody.)

[ELISA method (IgE antibody detection)]
1) Plate Coating with Rat Monoclonal Antibody Mouse IgE Antibody Rat monoclonal antibody against mouse IgE antibody, adjusted to a concentration of 4 μg / ml with 0.05 M carbonate buffer (pH 9.0), is added to 100 μl in a 96-well assay plate. Dispense per well and leave at 37 ° C. for 3 hours.
2) Blocking of the plate Bovine serum albumin (BSA) was dissolved in 0.2 M phosphate buffer (pH 7.2: PBS) at a concentration of 1 mg / ml, and dispensed at 100 μl / well into the plate of 1) above. Leave at room temperature for 1 hour.
3) Dilution and addition of serum sample (primary antibody) 10 times the serum of mouse immunized with aluminum hydroxide-antigen or phospholipid membrane preparation of the present invention in PBS (PBSA) containing BSA at a concentration of 1 mg / ml Beginning with dilution, dilute 11 times in 2-fold steps, dispense 50 μl / well into the plate in 2) above, and leave at room temperature for 1 hour.
4) Addition of biotinylated antigen After washing the plate in 3) above three times with PBS, dispense 100 μl / well of PBSA solution (1 μg / ml) of the biotinylated antigen solution and leave it at room temperature for 1 hour. .
5) Addition of peroxidase-labeled streptavidin solution After washing the plate in 4) three times with PBS, dispense 100 μl / well of peroxidase-labeled streptavidin solution and leave at room temperature for 1 hour.
6) Addition of enzyme substrate solution;
After washing the plate of 5) 3 times with PBS, o-phenylenediamine dihydrochloride (0.5 mg / ml) dissolved in citrate buffer was dispensed at 100 μl / well and allowed to stand at room temperature for 15 minutes. Develop color. After color development, the reaction is stopped by dispensing 2 M sulfuric acid in a volume of 50 μl / well.
7) Measurement using an absorptiometer;
Using an ELISA plate reader, the absorbance is measured at 490 nm, the end point of color development is determined, and the average dilution factor of the sample serum at that point is used as the ELISA titer (ELISA titer is used as the quantitative value of IgE). .

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example and a comparative example, this invention is not limited to a following example at all.

Example 1
(Preparation of liposome)
1) Preparation of lipid mixed powder 0.7560 g (1.2157 mmol) of didodecanoylphosphatidylcholine, 0.5287 g (0.9118 mmol) of didodecanoylphosphatidylethanolamine, 0.8223 g (2.1274 mmol) of cholesterol and didodecanoylphosphatidylserine 0.3927 g (0.6078 mmol) of Na salt was placed in an eggplant-shaped flask, and 50 ml of a mixed solvent of chloroform / methanol / water (65/25/4, volume ratio) was added and dissolved at 40 ° C. Next, the solvent was distilled off under reduced pressure using a rotary evaporator to form a lipid thin film. Further, 30 ml of distilled water for injection was added and stirred to obtain a uniform slurry. This slurry was frozen with liquid nitrogen and dried with a freeze dryer for 24 hours to obtain a lipid mixed powder.

2) Preparation of liposome Next, 60 ml of a separately prepared buffer solution (0.12 mM Na ₂ HPO ₄ , 0.88 mM KH ₂ PO ₄ , 0.25 M saccharose, pH 6.5, hereinafter abbreviated as “buffer solution”) The mixture was placed in an eggplant-shaped flask containing the lipid mixed powder, and the lipid was hydrated while stirring at 40 ° C. to obtain liposomes. Next, the particle size of the liposome was adjusted using an extruder. First, the obtained liposome was passed through an 8 μm polycarbonate filter, and then passed through the filter in the order of 5 μm, 3 μm, 1 μm, 0.65 μm, 0.4 μm and 0.2 μm. The average particle size of the liposome particles was 191 nm (measured by dynamic light scattering method).

(Administration immunity test with suspension of OVA-bound liposome)
1) Preparation of liposome preparation 2 ml of the obtained liposome was collected in a test tube, and 0.5 ml of ovalbumin (manufactured by Sigma, reagent, hereinafter sometimes referred to as “OVA”) solution (12 mg / ml) was added. . Next, 0.5 ml of a 2.4% glutaraldehyde solution was dropped, and then gently mixed for 1 hour on a 37 ° C. warm bath to immobilize ovalbumin on the outer aqueous phase side of the liposome. Next, 0.5 ml of 2M glycine-NaOH buffer (pH 7.2) was added, and the solution was left at 4 ° C. overnight to inactivate unreacted glutaraldehyde. Further, this solution was passed through a column packed with Sepharose CL-4B (Pharmacia Biotech, trade name) to fractionate the target product to obtain a liposome suspension in which the antigen was bound to the surface of the phospholipid membrane. The phosphorous concentration in the liposome suspension was measured (Phospholipid Test Wako), and the concentration was adjusted by diluting with a buffer solution so that the phosphorous concentration derived from phospholipid was 2 mM to obtain an OVA-bound liposome suspension. . Using the activation-labeled OVA, the same procedure as described above was performed separately, and the amount of OVA bound when the phosphorus concentration derived from the phospholipid of the liposome was 2 mM was 49 μg / ml.

2) Antibody production test BALB / c mice (female, 8 weeks old, 6 mice / group) were used, and 200 μl / dose of OVA-bound liposome suspension was intraperitoneally injected with a syringe, and after 4 weeks, the same method was used. Then, secondary immunization was performed. Serum was collected every week from the start of the test to 6 weeks later, and the changes in antibody titers (IgG and IgE) were measured by ELISA.

3) Measurement of IgG antibody by ELISA The IgG antibody titer was measured by the following method using mouse serum collected from the start of the immunization test up to 6 weeks.
a) Coating of plate with antigen Ovalbumin was dissolved in 0.05 M carbonate buffer (pH 9.0) at a concentration of 1 mg / ml, and dispensed into a 96-well assay plate in a volume of 50 μl / well for 1 hour at room temperature. I left it alone.
b) Blocking bovine serum albumin (hereinafter referred to as “BSA”) of the plate was dissolved in 0.2 M phosphate buffer (pH 7.2: hereinafter referred to as “PBS”) at a concentration of 1 mg / ml. The plate was dispensed at 100 μl / well and left at room temperature for 1 hour.
c) Dilution and addition of serum sample (primary antibody) Starting with 10-fold dilution of mouse serum immunized with antigen-bound liposome preparation in PBS containing BSA at 1 mg / ml concentration (hereinafter referred to as “PBSA”) Dilution was performed 11 times in stages, and 50 μl / well was dispensed into the plate of b) above, and left at room temperature for 1 hour.
d) Addition of peroxidase-labeled rabbit anti-mouse IgG antibody solution (secondary antibody) After washing the plate in c) three times with PBS, dispense 50 μl / well of PBSA solution of peroxidase-labeled rabbit anti-mouse IgG antibody. And left at room temperature for 1 hour.
e) Addition of enzyme substrate solution The plate of d) above was washed 3 times with PBS, dissolved in citrate buffer, and 100 μl / well of o-phenylenediamine dihydrochloride (0.5 mg / ml). The solution was dispensed and allowed to stand at room temperature for 15 minutes for color development. After color development, 2M sulfuric acid was dispensed at 50 μl / well to stop the reaction.
f) Measurement using an absorptiometer Using an ELISA plate reader, the absorbance was measured at 490 nm, the end point of color development was determined, and the dilution factor of the sample serum at that point was used as the ELISA titer (ELISA titer was IgG Used as a quantitative value of the antibody).

4) Measurement of IgE antibody by ELISA The IgE antibody titer was measured by the following method using mouse serum collected from the start of the immunization test to 6 weeks.
a) Coating of Plate with Rat Monoclonal Antibody Mouse IgE Antibody A rat monoclonal antibody against mouse IgE antibody, adjusted to a concentration of 4 μg / ml with 0.05 M carbonate buffer (pH 9.0), was added to a 96-well assay plate at 100 μl. / Well and dispensed at 37 ° C. for 3 hours.
b) Blocking of the plate Bovine serum albumin (BSA) was dissolved in 0.2 M phosphate buffer (pH 7.2: PBS) at a concentration of 1 mg / ml, and dispensed at 100 μl / well into the plate a) above. Left at room temperature for 1 hour.
c) Dilution and addition of serum sample (primary antibody) From 10-fold dilution of mouse serum immunized with aluminum hydroxide-antigen or liposome formulation of the present invention in PBS (PBSA) containing BSA at a concentration of 1 mg / ml For the first time, dilution was performed 11 times in a 2-fold step, and 50 μl / well was dispensed into the plate of b), and left at room temperature for 1 hour.
d) Addition of biotinylated antigen After washing the plate in c) three times with PBS, 100 μl / well of PBSA solution (1 μg / ml) of the biotinylated antigen solution was dispensed and allowed to stand at room temperature for 1 hour. .
e) Addition of peroxidase-labeled streptavidin solution The plate of d) above was washed three times with PBS, and then 100 μl / well of peroxidase-labeled streptavidin solution was dispensed and left at room temperature for 1 hour.
f) Addition of enzyme substrate solution After washing the plate in e) three times with PBS, o-phenylenediamine dihydrochloride (0.5 mg / ml) dissolved in citrate buffer was dispensed at 100 μl / well. The color was allowed to stand for 15 minutes at room temperature. After color development, 2M sulfuric acid was dispensed at 50 μl / well to stop the reaction.
g) Measurement using an absorptiometer;
Measurement was performed at 490 nm using an ELISA plate reader to determine the end point of color development, and the average dilution factor of the sample serum at that point was used as the ELISA titer (ELISA titer was used as the quantitative value of IgE antibody). .
5) antibody titer results;
Table 3 shows the antibody titers of IgG and IgE at 6 weeks and the IgE / IgG ratio calculated from the antibody titers.

(Example 8)
(Preparation of liposome)
1) Synthesis of a reactive phospholipid (succinimidyl-didecanoylphosphatidylethanolamine) composed of terminally modified phosphatidylethanolamine Dissolve and add 2 g of didecanoylphosphatidylethanolamine and 180 μl of triethylamine to a 4-neck with a capacity of 300 ml. Placed in flask. While stirring the inside of the flask at room temperature with a magnetic stirrer, a solution prepared by dissolving 3 g of disuccinimidyl succinate, which is a separately prepared bifunctional reactive compound, in 80 ml of chloroform was added over 4 hours according to a conventional method. The solution was added dropwise, and one end of disuccinimidyl succinate was reacted with the amino group of didecanoylphosphatidylethanolamine. This crude reaction solution was transferred to an eggplant flask, and the solvent was distilled off by an evaporator. Next, a small amount of chloroform capable of dissolving the crude reaction product was added to the flask to obtain a high concentration crude reaction solution, and silica gel equilibrated with chloroform / methanol / water (65/25/1, volume ratio) was used. Column chromatography is performed according to a conventional method, and only the fraction in which one end of disuccinimidyl succinate is bonded to the amino group of the desired didecanoylphosphatidylethanolamine is recovered, and the solvent is distilled off to remove the target reactive phosphorus. A lipid, succinimidyl-didecanoylphosphatidylethanolamine, was obtained.

2) Preparation of lipid mixed powder 0.0337 g (0.0541 mmol) of didodecanoyl phosphatidylcholine, 0.2165 g (0.2705 mmol) of succinimidyl-didecanoylphosphatidylethanolamine prepared in 1) above, 0.5021 g of cholesterol (1. 2986 mmol) and 1.7477 g (2.706 mmol) of didodecanoylphosphatidylserine Na salt in an eggplant type flask, and 50 ml of a mixed solvent of chloroform / methanol / water (65/25/4, volume ratio) was added at 40 ° C. Dissolved. Next, the solvent was distilled off under reduced pressure using a rotary evaporator to form a lipid thin film. Further, 30 ml of distilled water for injection was added and stirred to obtain a uniform slurry. This slurry was frozen with liquid nitrogen and dried with a freeze dryer for 24 hours to obtain a lipid mixed powder.
3) Preparation of liposomes Liposomes were prepared in the same manner as in Example 1, “2) Preparation of liposomes”. The average particle size of the liposome particles was 181 nm (measured by dynamic light scattering method).

(Administration immunity test with suspension of OVA-bound liposome)
1) Collect 1.5 ml of the obtained liposomes in a test tube, and add 3 ml of a separately prepared ovalbumin (Sigma, Reagent, hereinafter sometimes referred to as OVA) solution (1.25 mM, buffer solution). After that, the reaction was gently stirred at 5 ° C. for 48 hours. This reaction solution was subjected to gel filtration using Sepharose CL-4B equilibrated with a buffer solution according to a conventional method. Since the liposome fraction is cloudy, the target fraction can be easily confirmed, but it may be confirmed with a UV detector or the like. The phosphorous concentration in the obtained liposome suspension was measured (Phospholipid Test Wako), and the concentration of the phospholipid-derived phosphorous was diluted with a buffer solution so that the phosphorous concentration was 2 mM. Got. The amount of OVA bound when the phospholipid-derived phosphorus concentration was 2 mM was 38 μg / ml.
2) By the same method as in Example 1, antibody production test, measurement of IgG antibody by ELISA method, and measurement of IgE antibody by ELISA method were performed. Table 3 shows the antibody titers of IgG and IgE at 6 weeks and the IgE / IgG ratio calculated from the antibody titers.

Example 9
(Preparation of liposome)
1) Synthesis of Reactive Phospholipid Containing Terminal-Modified Phosphatidylethanolamine (Maleimide-didodecanoylphosphatidylethanolamine) Disacsin Imine in “1) Synthesis of Reactive Phospholipid Consisting of Terminal Modified Phosphatidylethanolamine” in Example 8 The number of moles of didodecanoyl phosphatidylethanolamine, triethylamine and bifunctional reactive compound used, and the subsequent procedures are the same except that the disuccinate was replaced with N-succinimidyl-4- (p-maleimidophenyl) propionate. To obtain the target reactive phospholipid, maleimide-didodecanoyl phosphatidylethanolamine.
2) Preparation of lipid mixed powder 1.0425 g (1.8428 mmol) of didecanoylphosphatidylcholine, 0.2375 g (0.3071 mmol) of maleimide-didodecanoylphosphatidylethanolamine prepared in 1) above, 0.8313 g of cholesterol (2. 1499mmol) and didecanoylphosphatidylglycerol Na salt 0.3888g (0.6143mmol) in an eggplant-shaped flask, and 50ml of a mixed solvent of chloroform / methanol / water (65/25/4, volume ratio) is added at 40 ° C. Dissolved. Next, the solvent was distilled off under reduced pressure using a rotary evaporator to form a lipid thin film. Further, 30 ml of distilled water for injection was added and stirred to obtain a uniform slurry. This slurry was frozen with liquid nitrogen and dried with a freeze dryer for 24 hours to obtain a lipid mixed powder.
3) Preparation of liposomes Liposomes were prepared in the same manner as in Example 1, “2) Preparation of liposomes”. The average particle size of the liposome particles was 165 nm (measured by dynamic light scattering method).

(Administration immunity test with suspension of OVA-bound liposome)
1) Collect 1.5 ml of the obtained liposomes in a test tube, and add 3 ml of a separately prepared ovalbumin (Sigma, reagent, hereinafter sometimes referred to as OVA) solution (1.25 mM, buffer solution). After that, the reaction was gently stirred at 5 ° C. for 48 hours. This reaction solution was subjected to gel filtration using Sepharose CL-4B equilibrated with a buffer solution according to a conventional method. Since the liposome fraction is cloudy, the target fraction can be easily confirmed, but it may be confirmed with a UV detector or the like. The phosphorous concentration in the obtained liposome suspension was measured (Phospholipid Test Wako), and the concentration of the phospholipid-derived phosphorous was diluted with a buffer solution so that the phosphorous concentration was 2 mM. Got. The amount of OVA bound when the phospholipid-derived phosphorus concentration was 2 mM was 40 μg / ml.
2) By the same method as in Example 1, antibody production test, measurement of IgG antibody by ELISA method, and measurement of IgE antibody by ELISA method were performed. Table 3 shows the antibody titers of IgG and IgE at 6 weeks and the IgE / IgG ratio calculated from the antibody titers.

(Examples 2-7 and 10)
(Preparation of liposomes and administration immunity test using suspension of OVA-bound liposomes)
1) According to each compounding molar ratio of phospholipid and cholesterol shown in Table 1, liposomes of Examples 2 to 7 and 10 were obtained by the same method as Example 1. Next, a suspension of each OVA-bound liposome was prepared by the same method as in Example 1 using the liposomes obtained in Examples 2 to 7 and 10.
The particle sizes (nm) of the liposomes obtained in Examples 2 to 7 and 10 were 188, 171, 149, 151, 154, 155, and 147, respectively (measured by dynamic light scattering method).
In addition, the binding amount (μg / ml) of OVA when the phospholipid-derived phosphorus concentration of the liposomes obtained in Examples 2 to 7 and 10 was 2 mM was 44, 46, 55, 57, 53, 51, respectively. , 51.
2) About the suspension of each OVA binding liposome obtained in Examples 2 to 7 and 10, by the same method as in Example 1, antibody production test, measurement of IgG antibody by ELISA method, and IgE antibody by ELISA method Measurements were made. Table 3 shows the antibody titers of IgG and IgE at 6 weeks and the IgE / IgG ratio calculated from the antibody titers.

  PC: diacylphosphatidylcholine, PS: diacylphosphatidylserine, PG: diacylphosphatidylglycerol, PE: diacylphosphatidylethanolamine, SI-PE: succinimidyl-diacylphosphatidylethanolamine, MI-PE: maleimide-diacylphosphatidylethanolamine

(Comparative Example 1)
1) Preparation of aluminum hydroxide gel suspension In a separately prepared buffer solution (1.2 mM Na ₂ HPO ₄ , 8.8 mM KH ₂ PO ₄ , pH 6.5), OVA was dissolved to 500 μg / ml, 1 ml of this OVA solution was added to 9 ml of an aluminum hydroxide gel (500 μg / ml) suspension prepared according to a conventional method to prepare an OVA-aluminum hydroxide gel suspension. The OVA concentration of this OVA-aluminum hydroxide gel suspension was 50 μg / ml.
(Administration immunity test of aluminum hydroxide gel suspension)
2) Antibody production test An antibody production test was carried out in the same manner as in Example 1 except that the OVA-aluminum hydroxide gel suspension prepared in 1) above was used instead of the liposome preparation of Example 1. Serum was collected every week from the start to 6 weeks later, and the changes in antibody titers (IgG and IgE) were measured by ELISA.
3) Measurement of IgG antibody by ELISA The IgG antibody titer was measured by the same procedure as in Example 1 using mouse serum collected from the start of the immunity test obtained in 2) above until the sixth week. The results are shown in Table 3.
4) Measurement of IgE antibody by ELISA The IgE antibody titer was measured by the same procedure as in Example 1 using mouse serum collected from the start of the immunity test obtained in 3) above until the sixth week. The results are shown in Table 3.
5) Results of antibody titer Table 3 shows the antibody titers of IgG and IgE at 6 weeks and the IgE / IgG ratio calculated from the antibody titers.

  PC: diacylphosphatidylcholine, PS: diacylphosphatidylserine, PG: diacylphosphatidylglycerol, PE: diacylphosphatidylethanolamine, SI-PE: succinimidyl-diacylphosphatidylethanolamine, MI-PE: maleimide-diacylphosphatidylethanolamine

(Comparative Examples 2-5 and 8)
(Preparation of liposomes, administration immunity test using suspension of OVA-bound liposomes)
1) According to the respective molar ratios of phospholipid and cholesterol shown in Table 2, liposomes of Comparative Examples 2 to 5 and 8 were obtained in the same manner as in Example 1. Next, a suspension of each OVA-bound liposome was prepared by the same method as in Example 1 using the liposomes of Comparative Examples 2 to 5 and 8.
The particle sizes (nm) of the liposomes obtained in Comparative Examples 2 to 5 and 8 were 263, 248, 242, 218, and 251 (measured by dynamic light scattering method), respectively.
In addition, the binding amount (μg / ml) of OVA when the phospholipid-derived phosphorus concentration of the liposomes obtained in Comparative Examples 2 to 5 and 8 was 2 mM was 45, 50, 53, 56, and 46, respectively. It was.
2) About the suspension of each OVA binding liposome obtained in Comparative Examples 2 to 5 and 8, by the same method as in Example 1, antibody production test, measurement by IgG antibody ELISA method and IgE antibody ELISA method Measurements were made. Table 3 shows the antibody titers of IgG and IgE at 6 weeks and the IgE / IgG ratio calculated from the antibody titers.

(Comparative Examples 6 and 7)
(Preparation of liposomes, administration immunity test using suspension of OVA-bound liposomes)
1) According to each compounding molar ratio of phospholipid and cholesterol shown in Table 2, liposomes of Comparative Examples 6 and 7 were obtained in the same manner as in Examples 8 and 9. Next, using the liposomes obtained in Comparative Examples 6 and 7, suspensions of OVA-bound liposomes were obtained in the same manner as in Examples 8 and 9.
The particle sizes (nm) of the liposomes obtained in Comparative Examples 6 and 7 were 252 and 255, respectively.
Moreover, the binding amount (μg / ml) of OVA when the phospholipid-derived phosphorus concentration of the liposomes obtained in Comparative Examples 6 and 7 was 2 mM was 39 and 38, respectively.
2) About the suspension of each OVA binding liposome obtained in Comparative Examples 6 and 7, by the same method as in Example 1, antibody production test, measurement of IgG antibody by ELISA method and measurement of IgE antibody by ELISA method went. Table 3 shows the antibody titers of IgG and IgE at 6 weeks and the IgE / IgG ratio calculated from the antibody titers.

In Table 3, each evaluation standard of the immunity test results is as follows.
1) Regarding IgG antibodies, Δ: 150 or more and less than 500, ○: 500 or more and less than 800, ：: 800 or more, and x: less than 150.
2) Regarding the IgE / IgG ratio, ◎; when 0.001 or less, ◯; when 0.001 or more and 0.005 or less, Δ; when 0.005 or more and 0.010 or less, ×; The case was over 0.010.
3) Comprehensive evaluation In the IgG antibody titer or IgE / IgG ratio, the comprehensive evaluation of those having one or more “x” or “Δ” evaluation was defined as “x”. Otherwise, the overall evaluation was “と し た”.

In the liposome preparations of Examples 1 to 10, it was confirmed that the production of IgG antibody was sufficiently enhanced from the IgG antibody titer, and the production of IgE antibody was suppressed from the IgE / IgG ratio. Moreover, it was confirmed that these performances were satisfied at the same time.
From this result, it was confirmed that the above-mentioned liposome preparation has an immune response regulating function. For this reason, the above-mentioned liposome preparation is suitable as a phospholipid membrane preparation used for vaccines or allergy treatment in which allergic symptoms are unlikely to occur.

Claims (10)

  An antigen or allergen is bound to the surface of a phospholipid membrane containing a phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms and a stabilizer for the phospholipid membrane; and A phospholipid membrane preparation, wherein the phospholipid contains diacylphosphatidylserine.   The phospholipid further contains at least one selected from diacylphosphatidylglycerol and diacylphosphatidic acid, diacylphosphatidylinositol, diacylphosphatidylcholine, diacylphosphatidylethanolamine, succinimidyl-diacylphosphatidylethanolamine, and maleimide-diacylphosphatidylethanolamine. The phospholipid membrane preparation according to claim 1, wherein   The phospholipid membrane preparation according to claim 1, comprising a lipid to which an antigen or allergen is bound by ionic bond or covalent bond.   The phospholipid membrane preparation according to any one of claims 1 to 3, wherein the stabilizer is cholesterol.   The phospholipid membrane preparation according to any one of claims 1 to 4, wherein the phospholipid membrane preparation is a liposome preparation.   1 to 99.6 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 0.2 to 75 mol% of a stabilizer for phospholipid membrane, and an acyl group having 10 to 12 carbon atoms Or 0.2 to 80 mol% of a lipid having a hydrocarbon group and bound to an antigen or allergen, and the acidic phospholipid contains diacylphosphatidylserine. Formulation.   1 to 99.5 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 0.2 to 75 mol% of a stabilizer for phospholipid membrane, and an acyl group having 10 to 12 carbon atoms Alternatively, 0.2 to 80 mol% of a lipid having a hydrocarbon group and having an antigen or allergen bound thereto, and 0.1 to 70 mol% of a neutral phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms And the acidic phospholipid contains diacylphosphatidylserine. A phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms , a lipid having a functional group capable of binding an antigen or allergen to the surface, and a phospholipid membrane stabilizer. And the phospholipid contains diacylphosphatidylserine. 1 to 99.6 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 0.2 to 75 mol% of a stabilizer for phospholipid membrane, and an acyl group having 10 to 12 carbon atoms or have a hydrocarbon group, a lipid 0.2 to 80 mol% having a functional group capable of binding the antigen or allergen to the surface, it comprising a and the acidic phospholipid and diacyl phosphatidyl serine A phospholipid membrane characterized by containing. 1 to 99.5 mol% of acidic phospholipid having an acyl group or hydrocarbon group having 10 to 12 carbon atoms, 0.2 to 75 mol% of a stabilizer for phospholipid membrane, and an acyl group having 10 to 12 carbon atoms or have a hydrocarbon group, a neutral phospholipid having a lipid 0.2 to 80 mol% having a functional group capable of binding the antigen or allergen to the surface, an acyl group or a hydrocarbon group having 10 to 12 carbon atoms A phospholipid membrane comprising 0.1 to 70 mol% of a lipid, and wherein the acidic phospholipid contains diacylphosphatidylserine.